Biguanide salts of penicillin



latented Mar. 10, 1953 2,631,146 BIGUANIDE SALTS or PENICILLIN Joseph F.Weidenheimer, Pearl River, and Lawrence Ritter, Valley Cottage, N. Y.,assignors to American Cyanamid Company, New York, N. Y., a corporationof Maine No Drawing.

9 Claims. 1

This invention relates to a new therapeutic composition which has as anessential ingredient a comparatively insoluble organic penicillin salt.More particularly the invention relates to organic salts of penicillinwhich areless soluble than procaine penicillin and which are non-toxicwhen injected whereby these compounds may be used for penicillintherapy, giving a blood level which is therapeutically effective over aprolonged period.

This application is a continuation-in-part of our application Serial No.87,826 filed April 15, 1949, entitled Penicillin Derivatives, and of ourapplication, Serial No. 268,933, filed January 29, 1952, entitledPenicillin Salts, now both abandoned in favor of this application.

' It is found that'thecompounds of this invention may readily be usedfor the injection of patients with such a quantity that atherapeutically useful concentration of penicillin is maintained in theblood of a normal patient for a period in excess of 48 hours so thatadditional injections are not required.

The therapeutic composition of this invention has as an essentialelement thereof a salt of penicillin with a blguanide or substitutedbiguanide which forms with the penicillin anon-toxic salt of suchsolubility characteristics that the material when administered to asubject is therapeutically available at such a rate as to build upsuificient penicillin concentration to be effective against theparticular organism for which the treatment is being given but yet whichis not sufficiently soluble to permit all of the penicillin to bedissipated within a comparatively short period.

The biguanide salts of penicillin of this application may also be usedin processes of refining penicillin as they form salts by whichpenicillin may be precipitated from solutions containing the penicillin.Also, certain of these salts are extremely insoluble and stable underadverse conditions so that the salts may be used as additives forfoodstufis where the penicillin salt is distributed and whereconventional salts or penicillin would be inactivated due to moisturepresent.

Among the biguanides, the para-halophenylbiguanides, the -'-nitrogen ofwhich may be substituted by one or more 'alkyl groups, have been foundto be particularly eflicacious. The prepae ration and certaincharacteristics of some of these biguanides are disclosed and set forthin a U. S. patent toDaniel E. Nagy, No. 2,455,896 entitled l-Aryl,5-Alkyl-Biguanides a British Application November 13, 1952, Serial No.320,334

Patent 577,843 to Francis H. S. Curd and Francis L. Rose, entitledBiguanide Derivatives; a U. S. patent to Francis H. S. Curd and FrancisL. Rose, 2,467,371, entitled Biguanicle Derivatives and a U. S. patentto Daniel E. Nagy, 2,455,897, entitled 1,5-Diarylbiguanides fromAromatic Amines and Dicyanimide.

Certain of the biguanide penicillins are sufiiciently heat stable thatthe salts may be sterilized by the use of heat, as for example, C. for10 hours, thereby enabling the sterilization of the penicillin salts ata later and more convenient stage in their manufacture.

Any of the penicillins may be used in this invention. At present themedical" profession prefers penicillin G, and accordingly the penicillinG salts are those which are normally prepared. From the standpoint ofconvenience and preparation, the availability of intermediates and thecomparatively low cost of the materiaLthe l-(pchlorophenyl) -5 isopropylbiguanide compound of penicillin G is among those which are normallycommercially preferable.

The chemistry appears to be that of the straight reaction between anacid and a base. Penicillin forms alkaline salts very readily and theherein used biguanides from acid salts.

As a method of preparation it is normally most convenient to mix analkali, or alkaline earth metal, or a suitable organic base, salt of thede-' sired variety of penicillin with a calculated quantity 0r slightexcess of an acid salt of the desired biguanide thereby forming thedesired complex. From the standpoint of cost and availability the saltsmost commonly used are the sodium, potassium, calcium or triethylaminesalts of penicillin and a halide, usually hydrochloride, salt of thebiguanide such as l-(p-chlorophenyl) -5 isopropyl biguanidehydrochloride. There is thereby formed the desired salt, and, sodium,calcium, potassium or triethylamine halide, such as chloride, whichmaterial is readily removed from the desired biguanide penicillin,although no harm is done if such a salt is injected with the penicillin.It may be that some type of complex other than that classed as a truesalt is formed by the interaction of the specific'biguanides andpenicillins herein mentioned, but for. purposes of con-' venience thereaction product will be referred to as a salt because it exhibitscertain of the properties of saltsand probably is properly classed as asalt.

In the preparation of these salts the initial crystallization at timesrequires considerable stirring and cooling; but normally the crystal 3habit is such that the crystals as formed are suitable for injection. Insome instances recrystallization from suitable solvents may be desired.

For purposes of injection it is desired that the particular biguanidepenicillin salt be suspended in a suitable medium such as distilledwater, normal saline, 20% propylene glycol or other commonly usedaqueous diluent or in an oil such as peanut oil, sesame oil, cottonseedoil or other assimilable triglyceride, together with such stabilizingagents, buiiers, thixatropic agents, viscosity modifiers and wettingagents as may be desired to cause the material to have a fluidit'y andstability such that it may be easily filledinto a hypodermic syringe andinjected into a patient.

Additionally, it is frequently desirable to have mixed with this solublepenicillin salt a "water soluble penicillin type such as the sodium orpotassium or calcium salt which will cause a high initial blood level ofpenicillin in a patient so that by the combination the patient receivesa very rapid, high blood level caused by th soluble penicillinderivative and there is maintained in the patient a therapeutic level ofpenicillin over a prolonged period because of the low solubility andlowered rates of absorption of our specific biguanide derivatives of thepenicillin.

Qertain of the biguanide derivatives of penicillin, particularly l(p-chlorophenyl) -5 isopropyl biguanide penicillin, may be used in lieuof procaine penicillin-in the various forms of ointments and suspendingmedia now used for procaine penicillin and thereby will be obtained aproduct possessing superior stability and a greater period oftherapeutic efficacy. I

In certain instances the 1-(p-chlorophenyl) -5 isopropyl biguanide willbe iound to have a therapeutic effect of its own although normallypatients requiring penicillin do not require treatment withl-(p-chlorophenyl) -5-isopropyl biguahide. The concentration ofl-(p-chlorophenyl) 5 isopropyl biguanide which is built up in thepatient is not sufliciently high to cause any complications and is lessthan that which is frequently used as a prophylactic in malaria control.

In some instances for patients who are particularly sensitive there maybe-added to the mixture a-local anesthetic to prevent pain at the pointof injection.

Becauseof -the:greater 'insolubility in water, if l-(p-chlorophenyl) -5isopropyl biguanide is added to azprocaine penicillin solution therewill be formed a I-(p chlorophenyD-k'; isopro'pyl biguanide penicillinand free procaine which may be as the hydrochloride. It may beconvenient to have procaine or other anesthetic present either from thisor other sources so that the sensitivity at the point of injection isdiminished.

Whereas our compounds may be formed by the interaction of salts ofpenicillin and salts of'the specific biguanides, whichare to be used,they may also be-forfned by the reaction of free penicillin and the"free biguanide. They may be formedi'n-a watersolution, or in an'aqueousmediumsuch as a mixture of water and alcohol, in analcohol mediumcontaining a large proporti'on'of alcohol, in water, or even ananhydrous alcohol, or any or the various organic solvents in "which theparticular biguanides and penicillins are each somewhat soluble and thesolubility characteristics of all of the compounds are such that/thedesiredproducts are formed thereby, even, for-example, by-removal of thesolvent 4 or other methods well known to those desiring to manufacturesalts.

More particularly, our invention includes the salts of penicillin suchas penicillin G F. X and other penicillins, hereinafter calledpenicillin, with a basic compound from the group consisting of biguanideand compounds containing a biguanide nucleus of the formula in R;

where R1, R2, R3 and R4 may be either hydrogen or such radicals asalkyl, alkenyl, alkynyl, cycloalkyl, monocycli'c aryl, polycyclic aryl,monocyclic aroyl, 5 membered monocyclic heterocyclic radicals and 6'membered monocyclic heterocyclic radicals, which radicals may either bethe simple radicals or substituted. It is found that such radicals asare mentioned above may have on the aliphatic portions such substituentsas :hydroxy, alkoxy, aryl and nitrile groups and on the aromaticportions such groups as alkyl, aryi, halogen, nitro, alkoxy, aryloxy,nitrile, hydroxy and carbethoxy groups. We prefer that not more than twoof these substituent groups be aromatic radicals as otherwise thebasicityof the biguanide is less than is preferred for the formation ofthe biguanide salts. For penicillin salt formation R5 and Rs may beeither hydrogen or lower alkyl groups, but it is normally moreconvenient to prepare the biguanides where R5 and Rs representhydrogens. Where R2 is hydrogen, R1 and R5 are equivalent, as thehydrogen is labile. Similarly, where R4 is hydrogen, R3 and Rs areequivalent.

To list all biguanides with which our salts may be formed would undulylengthen this disclosure, but by way of example, certain typical onesare listed. Among the new salts of our invention are the salt ofpenicillin with:

i-ethylbiguanide 1,1-diethylbiguanide 1,2-diethylbi'guanide 1gfi-diethylbiguanide l -ethyl -'5 -propylbiguanide 1,1-diethyl-2ethylbiguanide 1,1-diethyl-5-ethylbiguanide 1 ,1,2 triethyl-5 propylbiguanide 1 beta-hydroxyethylbiguanide l -'beta methoxyethylbiguanide1,1 -"dibutylbiguanide 1 -betacyanoethylbiguanide 1-betaorthochlorophenyl) -ethylbiguanld 1-beta-phenylethylbiguanidephenylbiguanide o-tolylbiguanide 1-octadecyl 5-phenylbiguanide1-butyL5-octylbiguanide l -benzenesulfony1-5 phenylbiguanide 1-'butyl-5, phenylbiguanide 1,'4-diphenyl-'3;5-diisopropylbiguanideLB-diphenylbiguanide l,5 di p chlorophenylbiguanide 1dodecyl-5-phenylbiguanide 1-dodecyl-5-p-sulfonamidobiguanide1,1-dibutyl-5-phenylbiguanide 1,5-dimethyl-Lfi-diphenylbiguanide 1,5-dibeta-naphthylbiguanide 1,5-di-o-chlorophenylbiguanide 1,5-"di--mchlorophenylbiguanide 1,5-di-p-methoxyphenylbiguanide 1 ,5di-p-bromophenylbiguanide 1,5-dip-nitrophenylbiguanide 1- (o'-biphenyl)biguanide 1 m-methoxyphenylbiguanide 1-m-phenoxyphenylbiguanidel-m-cyanophenylbiguanide l-p-hydroxyphenylbiguanidel-p-carbethoxyphenylbiguanide 1,1-dibutyl-5-methyl-5-phenylbiguanide1-butyl-5-methyl-5-m-tolylbiguanide1,1-di-betaehydroxyethyl-5-p-tolylbiguanide1-allyl-5-alpha-naphthylbiguanide l n hexyl 5ethyl-5-p-tert-amylphenylbi-.

guanide 1-tert-amyl-5eoeanisylbiguanide1.1epentamethylenee5-p-nitrophenylbiguanide1-cyclohexyl-Beethyl-5-p-chlorophenylb-iguanide1,1+diallyl-5ep-cyanophenylbiguanide 1;5,5-tributylbiguanide L Any ofthe above listed penicillin salts may be formed by reactionof-penicillin and the biguanide. As illustrative examples showing theproduction of suchsalts'in accordance with this invention are: c l

EXAMPLE 1 p In a mixture of 2500 milliliters of distilled water and 750milliliters of ethyl alcohol were dissolved 125 grams of l-(p--chlorophenyll-5 iso propyl biguanide acetate. In an equal volume of asimilar solution were dissolved 175 grams of the triethylamine salt ofpenicillin. The l-(pchlorophenyl)- 5 isopropyl biguanide acetatesolution was "filtered through a sterilizing filter into a previouslysterilized reaction vessel equipped with mechanical stirrer, inlet tubesand a. filter foot. The triethylamine penicillin was sterile filteredand slowly introduced into the reaction vessel with stirring. Afterabout of the penicillin solution had been introduced, the'1'e' actionmixture wassterilely seeded to induce crystallization. The'remainder ofthe penicillin solution was added over a period of about 1 hours, thel-(p-ohlorophenyD-5 isopropyl bi: guanide Penicillin .crystallizing asformed. The mixture was 'placed'in'the chill room at approximately C.for two hours to insure complete precipitation. "The solvent was removedthrough the filter foot and the residual l-(p-chlorophenyl) isopropylbiguanide .penicillin crystals washed with approximately 1 liter. ofsterile .dis-' tilled water anddried for 48 hours. The entire:

procedure, after the sterilefiltrations, was carried out; under;Sterile; conditions, so that the material as formed was sterile. Therewas obtained a yield of 163 grams-or 69% of the theoretical yield.Theoretically the material should assay 1015 units per milligram; onactual bioanalysis it was found to show 1043 units per milligram. Thel-(p-chlorophenyD-S isopropyl biguanide penicillin as thus formed wasfound to have a melting point of approximately 161-162 C. and was foundto be soluble tothe extent of approximately 0.257% in water. 200milligrams of, the thus obtained l-j(p-chlorophenyl)-5 iso propylbiguanide penicillin dry was mixed with sufiicient sesame oil to form 1cc. of a suspension,

thereby forming an injectable therapeuticgdose EXNEAPLE 2 To a solutionof 125 grams of l-(p-chloro phenyl)-5 isopropyl biguanide acetate in2500.

milliliters ofjdistilled water and 750 milliliters,

of alcohol was added slownwitustirrinea some; 1 mixedwit a n rmalsalineiiiluent. a. therapeutic.

6 tion-of 137 Tgrams of sodium'penicillinin 1000 milliliters ofdistilled water. After approxi mately of the penicillin had been run in,the addition was stopped and the material seeded withvl-(p-chlcrophenyl)-5 isopropyl biguanide penicillin crystals. As thesolution became opalescent indicating that crystallization hadcommenced, theremainder of the penicillin was added over a period ofapproximately l hours with continuous stirring. Strict conditions ofcleanliness were maintained but the materials were not sterile norsterilized. The suspension was placed in a chill room at approximately 0C. overnight to allow complete precipitation, and the solvent removed.The residual crystals of 1-'(p-chlorophenyl)--5 isopropyl biguanidepenicillin were washed with distilled water and dried at-roomtemperature in a desiccator for 48 hours givinga yield of 195 grams or87% of the theoretical amount. Thel--(p-chlorophenyl)-5'isopropyl-biguanide penicillin was filled intoampoules containing the desired dosages and the ampoules were sterilizedin an oven at C. for 3 hours. Thereby were obtained sterile ampoulescontaining dry 1-(p-chlorophenyD-5 isopropyl biguanide penicillin inthedesired dosage units. For injection, to such an ampoule may be addeda sufficient quantity of a sterile aqueous 20% propylene glycol diluentto form a suspension having the desired concentration for injection intothe patient.

EXAMPLE 3 7 116 grams of l-(p-chlorophenyD-S isopropyl biguanidehydrochloride were dissolved in a solution of 2500 milliliters ofdistilled water and 750 milliliters of alcohol. Thereto was added gramsof potassium penicillin G in a solution of 2500milliliters of distilledwater and 750' millilit'ers of alcohoL- The penicillin was added slowlywith stirring; the mixture being seeded to insure crystallization, afterpart of the penicillin-containing solutionwas added.- The mixture wasstirred thoroughly and permitted to stand overnight! The crystals wereremoved from the solvent thereby being obtained l-(p-chlorophenyl)-5isopropyl biguanide penicillin in an unsterile condition. The thusprepared l-(pchlorophenyD-5 isopropyl biguanide penicillin wassterilized by heating to 110 C. for 10 hours; after which it was readyfor blending, mixing and filling under sterile conditions fortherapeutic EXAMPLE4 '125 grams of 1-(p-chlorophenyll-5 isopropylbiguanide acetate was powdered and suspended in 1 liter of distilledwater. Thereto was added a solution of 1'75 gramsoftriethylaminepenicillin dissolved in 1 liter of water. The suspensionwas stirred for 5 hours with a small quantity of crystalline1-(p-chlorophenyl) -5 isopropyl biguanide penicillin added as seedduring the first hour. The desired 1- (p-chlorophenyl )-5 iso propylbiguanide penicillin was separatedfrom the aqueous layer, washed withwater to remove salt -anddried under high vacuum for 48'hours. The driedmaterial was sterilizedby heating to 110 C. for 8 hours. The thusprepared dry sterile l-(p-chlorophenyD-B isopropyl biguanide penicillinwasmixed with sterile potassium penicillin and the mixture filledintovials, each vial containing a sufi'icient quantity of; each of the 1-(p-cholorop-henyll-B isopropyl biguanide penin. and potassium. pen c l nto prov de. wh

7 dose giving high initial levels and prolonged -levels in accordancewith the desire of the user.

EXAMPLE '5 5 grams of 1-(p-chlorophenyll-5 isopropyl biguanide acetatewere dissolved ina mixture of 100 milliliters of water and 30milliliters of ethyl a1- cohol. Thereto were added 7.15 grams of thetriethylamine salt of penicillin G dissolved in 100 milliliters of waterand .30 milliliters of alcohol over a period of one hour. A very smallquantity of seed crystals were added, the crystallization beginningalmost immediately, and at the end of the mixture was quite pasty. Themixture was stirred for an additional hour at C., the crystalscollected, washed in distilled water, .anddried in vacuo. There wasobtained a yield of 86% of the theoretical analyzing 1040 units permilligram and melting at 161-162 C. and showing an EXAMPLE 6 960 gramsof para-chlorophenyl-biguanide hydrochloride dissolved in a mixture of25 liters of water and liters of ethyl alcohol were treated with 2.2kilograms of the triethylamine salt of penicillin dissolved in 25 litersof water and 5 liters of alcohol. The penicillin solution was added tothe biguanide solution and when an opalescent solution was obtained, themixture was seeded and stirred until crystallization began, theremainder of the penicillin solution then being slowly added. Thecrystals formed, clusters of thin rods, were collected, washed withwater and dried in vacuo. There was obtained a yield of 63% of thetheoretical; the crystals melted .at 12l-l22 C. (uncorrected), thematerial was soluble to the extent of 0.3% in water. Because thecrystals melt so close to the temperatures :required for heatsterilization, as a matter of convenience the product is prepared understerile conditions .to obviate the necessity of heat sterilization afterits preparation. The material was found to have an activity of 1079units per milligram, and on analysis showed:

Percent Carbon 52.180 Hydrogen 5.14 Nitrogen 17.99

EXAMPLE 7 EXAMPLE 8 1 gram of 1,1-dibutyl-5-(para-iodophenylibiguanidehydrochloride was dissolved in milliliters of water and 15milliliters-of alcohol. The clear solution was'then treated with asolution of 1 gram of potassium penicillin dissolved in a mixture of17.5 milliliters of water and "7.5

milliliters of alcohol. An oil was formed which upon storage overnightat 4 C. yielded crystals in hexagonal plates which were collected, driedand found to assay 751 units per milligram.

EXAMPLE 9 Phenylbiguanide penicillin 1 gram of phenylbiguanidehydrochloride dissolved in 20 milliliters of water is added withstirring :to a solution of 2 grams of triethylamine penicillin Gdissolved in 20 milliliters vof water. An oil is deposited which doesnot crystallize readily on standing. The supernatant liquid is decantedto yield a gummy residue which upon drying in a vacuum oven yields :adry semicrystalline powder. The power analyzes-917 units per milligram.

EXAMPLE o-iolylbiyu uide penicillin 1 gram of o-tolylbiguanidehydrochloride dissolved in 20 milliliters of water is added withstirring to a solution of 1.9 grams of triethylamine penicillin G in 20milliliters of water. A gum is deposited which upon drying in a vacuumoven yields a dry powder having a potency of 9.30 units per milligram.

EX M E.

1-octaclecyl-S-phenylbiguanide penicillin A solution is prepared'of 0.5gram of l-octadecyl-fi-phenylbiguanide hydrochloride, as the free base,in 15 milliliters of ether. A solution of 0.4 gram of the free acid formof penicillin (penicillinic acid) in '30 milliliters of ether is addedwith stirring. The resulting clear ethereal solution is evaporated todryness, yielding the amorphous l1octadecyl-5-phenylbiguanidepenicillin.

EXAMPLE 1'2 1,5-dzfphcnylbiyuanide penicillin A solution is prepared of4.1 grams of 1,5-dlphenylbiguanide hydrochloride dissolved in a mixtureof 50 milliliters of ethyl alcohol and 25 milliliters of water. Thissolution is added to a solution of 5 grams of sodium penicillin in 25milliliters of water. The combined mixtures are stirred and on cooling,the desired L-S-diphenylbiguanide penicillin salt separates as crystals.

EXAIVIPLE 13 1,5-di p-chlorophenylbi uanide penicillin .5igra-ms of1,5-di-p-chlorophenylbiguanide hydrochloride is suspended in '25milliliters of ethyl alcohol and 50 milliliters of water. To this isadded dropwise with stirring a solution containing '5 grams ofcrystalline sodium penicillin G in the same solvent. The biguanidehydrochloride dissolvesand the desired 1,5-di-p-chlorophenylbiguanidepenicillin precipitates in crystalline form. The precipitate iscollected, washed with water, and dried. The yield is 8.7 grams ofmaterial assaying 861 units per milligram. The melting point isapproximately "161 C. and the material is soluble to the extent of0.01459; in water. The low solubility ,makes this salt particularlyuseful as an additive for poultry feed. A sampleof this salt, upon beingheated at 110 C. for 11 hours, is found to have a potency of 833 unitsper milligram, showing that the salt 'may beheatsterilized.

9 EXAMPLE 14 1,5-di-alpha-naphthylbiguanide penicillin grams of1,5-di-alpha-naphthylbiguanide hydrochloride is mixed with 50milliliters of alcohol and 50 milliliters of water. Thereto is added 5grams of sodium penicillin in 25 milliliters of water. On cooling andstanding, the amorphous 1,5-di-alpha-naphthylbiguanide pencillinseparates out.

Other biguanides acid salts such as the sulfate, the nitrate, thephosphate or the acetate may be used instead of the hydrochloride salts.A wide variety of salts of penicillin may be used but the alkali orammonium or alkylamine salts are particularly convenient. The reactionmay be carried out in water, methanol, ethanol, propanol, butanol,2-ethoxyethanol, 2-methoxyethanol, other alkoxy ethanols, otheralcohols, dioxane, formamide, dimethylformamide, acetone, methyl ethylketone, or methyl isobutyl ketone, or mixtures of two or more of thesesolvents or other similar solvents.

Having thus set forth by description and example certain aspectsthereof, as our invention we claim:

1. The salt of penicillin and a compound of the formula where R1represents a member selected from the group consisting of alkyl,monocyclic aryl, halomonocyclic aryl, and dicyclic aryl radicals; R2represents a member selected from the group consisting of hydrogen,alkyl, monocyclic aryl, chloromonocyclic aryl, and dicyclic arylradicals; and R3 represents a member selected from the group consistingof hydrogen and alkyl radicals.

2. A salt of a penicillin and a compound of the formula No referencescited.

1. THE SALT OF PENICILLIN AND A COMPOUND OF THE FORMULA